DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof

An infectious cloning, encephalitis virus technology, applied in the field of reverse genetics technology and RNA virus rescue, can solve problems that have not yet been seen, and achieve good infectivity

Inactive Publication Date: 2013-05-08
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] At present, there are no domestic and foreign reports on the construction of D

Method used

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  • DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof
  • DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof
  • DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain, as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0048] Example 1 Identification of the genome sequence of Japanese encephalitis virus vaccine strain SA14-14-2

[0049] 1. Obtaining of SA14-14-2 strain virus genomic RNA from live attenuated vaccine

[0050] ⑴ Take a lyophilized preparation of Japanese encephalitis live attenuated vaccine (batch number 200710049-2, SA14-14-2 virus content: 5.4lgPFU), add 0.5mL virus diluent for reconstitution.

[0051] (2) Extraction of viral genomic RNA: add 0.8ml Trizol reagent to 0.5ml vaccine diluent, mix well, and stand still at room temperature for 5 minutes; add 0.2ml chloroform, mix well, and stand still at room temperature for 5 minutes; centrifuge at 4°C, 12000g×20min.

[0052] (3) Take the upper water phase, transfer it into a new centrifuge tube without RNase pollution, add an equal volume of isopropanol for extraction, and let it stand at room temperature for 10 minutes. Centrifuge at 4°C, 12000g×20min.

[0053] ⑷ Discard the supernatant, add 0.5ml of 75% alcohol to rinse, mix well and s...

Example Embodiment

[0066] Example 2 Transformation of RNA-based JEV infectious clones into DNA-based JEV infectious clones

[0067] 1. Modification of 5` Semi-molecular Clones of SA14-14-2 Vaccine Strain Genome

[0068] Plasmid pBRKpn-J1J2 (Ying, H., 2003) is a 5'half molecular clone of SA14-14-2 vaccine strain, including the genome sequence of 1-5581 nucleotides. On this basis, the CMV sequence was transformed by fusion PCR technology (SEQ ID NO: 27) and SA14-14-2 genome 5'end sequence, cloned into plasmid vector pBRKpn-J1J2 through MluI and ClaI restriction sites, and further use fusion PCR technology to combine the two chimeric introns The sequence (SEQ ID NO: 28) was inserted into JEV genome 356 and 2217 to construct plasmid pCMV-J1J2. In addition, according to the sequencing results in Table 1, site-directed backmutation was performed on individual nucleotide mutations in plasmid pCMV-J1J2.

[0069] 2. Modification of 3'half molecular clone of SA14-14-2 vaccine strain genome

[0070] ⑴Add geneti...

Example Embodiment

[0083] Example 3 Evaluation of the infectivity of SA-14-14-2 DNA-based full-length clone pQSINGZ at the cellular level

[0084] In order to evaluate the infectivity of SA-14-14-2 DNA-based full-length clones in vitro, the purified plasmid was transfected into BHK-21 cells to observe whether the virus production was restored.

[0085] ⑴ Purification and transfection of pQSINGZ-EG

[0086] Use large plasmid kit (Plasmid Max Kit, Qiagen company) to extract and purify the SA14-14-2 full-length cloned plasmid pQSINGZ-EG containing the reporter gene EGFP, and adjust the plasmid concentration to 1μG / μL. Lipofectamine TM 2000, Invitrogen company) method transfected BHK-21 cells, every 24 hours after transfection, observe and record under a fluorescence microscope ( Image 6 ).

[0087] ⑵PQSINGZ purification and transfection

[0088] Use large plasmid kit (Plasmid Max Kit, Qiagen company) to extract and purify the SA14-14-2 full-length cloned plasmid pQSINGZ, adjust the plasmid concentration ...

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Abstract

The invention relates to a DNA (Deoxyribose Nucleic Acid)-based infectious clone of a Japanese encephalitis virus SA14-14-2 strain and a construction method of the infectious clone. The infectious clone is constructed by adopting a pBR322 plasmid as a framework vector and then inserting the full-length cDNA of the Japanese encephalitis virus SA14-14-2 vaccine strain; the 5' end of the full-length cDNA of the SA214-14-2 vaccine strain is connected with a CMV (Cucumber Mosaic Virus) promoter, a BGH (Bovine Growth Hormone) polyadenylation sequence is added at the 3' end, and gomphosis intron sequences for stabilizing are respectively added on the 356 locus and the 2217 locus of the genome cDNA. The invention also provides application of infectious clone serving as a novel viral vector, and thus a solid foundation is provided for developing a plurality of novel vaccines for preventing and treating tumours and viral diseases.

Description

technical field [0001] The invention relates to reverse genetics technology and RNA virus rescue technology, in particular to the DNA-based infectious clone of Japanese encephalitis virus SA14-14-2 strain, its construction method and application. Background technique [0002] Yellow fever viruses belong to positive-strand RNA viruses. This genus contains more than 80 members, and more than half of them can induce diseases in humans and animals. It poses a great threat to human health (Monath TP, 1996), such as JEV, dengue fever virus (DEN), yellow fever virus (YFV), tick-borne encephalitis virus (TBEV) and so on. For a long time, the development of related vaccines and drugs has been very slow. So far, only YFV, JEV, and TBEV have vaccines, while most of the other members have no effective prevention and treatment methods. [0003] Epidemic Japanese encephalitis (JE) is the most common type of viral encephalitis in Asia. my country is the country with the largest number of ...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66C12N15/86C12N7/00C12N7/01A61K39/12A61K39/295A61P31/14A61P35/00C12R1/93
Inventor 黄莺贾丽丽孙志伟徐宏山俞炜源俞永新
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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